1. Field of the invention
The invention relates to a liposomal formulation containing a therapeutic agent. Moreover, it relates to an original liposomal formulation allowing a modulated release of the therapeutic agent over time, as well as an increased penetration of a therapeutic agent such as an antibiotic into bacterial cells. The invention further relates to a method of treating bacterial infections in an animal through the administration of the formulation of the present invention.
2. Description of the prior art
Encapsulation of bioactive compounds in natural or synthetic matrixes has been extensively studied over the past decades. Advantages of such strategy of administration are numerous. First, it provides a protection from the inactivation or degradation of the bioactive compound. Secondly, it controls the kinetics of compound release, allowing the optimization of the blood concentration profile. This diminishes the deleterious effects of bioactive compounds with short half lives. In addition, it permits a reduction of the risk of toxicity.
Liposomes are closed microscopic vesicles that form spontaneously from phospholipids above their transition temperature, in the presence of excess water. Vesicles with a diameter ranging from 20 nanometers to several micrometers can be prepared. Multilamellar liposomes are made of concentric phospholipid bilayers separated by aqueous layers. Unilamellar liposomes consist of a single phospholipid layer surrounding an aqueous core. Liposomes can accommodate hydrophilic molecules in the aqueous spaces and lipophilic molecules in the lipid bilayers.
The potential of liposomes as vehicles for therapeutic agents, or therapeutic liposomal formulations, has been studied by several investigators. Successful treatments with liposomes against intracellular bacteria have been demonstrated (Lopez-Berestein et al., 1987, J. Clin. Oncology, 5:310-317; and Popescu et al., 1991, U.S. Pat. No. 4,981,692). A number of studies have also shown that liposome-entrapped antibacterial agents increase the therapeutic indices of these agents as a result of decreased toxicity, modification of pharmacokinetics and tissue distribution parameters (Lagace et al., 1991 J. Microencapsulation 8:53-61 and references therein; Omri et al., 1994, Antimicrob. Agents Chemother. 38:1090-1095).
The most widely used type of antibacterial agent is most certainly the antibiotics. Antibiotics can be subdivided in different groups which include the .beta.-lactams, aminoglycosides, macrolides, lincomycin, clindamycin, tetracyclines, chloramphenicol, vancomycin, rifampin, quinolones, and sulfonamides.
Aminoglycosides are all potent bactericidal agents that share the same general range of antibacterial activity and pharmacokinetic behaviour. The members of the group are typified by the presence of aminosugars glycosidically linked to aminocyclitols. The main agents fall into two groups: the small group consisting of streptomycin, and its close relatives; and the large group which is subdivided into the neomycin group, the kanamycin group which is again subdivided into the kanamycins, tobramycin and their semi-synthetic derivatives amikacin and dibekacin and the important sub-group of gentamicins and their relatives, netilmicin and sissomicin.
The aminoglycosides inhibit protein synthesis in a variety of microorganisms and are used primarily to treat infections caused by organisms which are resistant to other antibiotics, particularly gram-negative bacteria such as but not limited to species of Escherichia, Enterobacter, Klebsiella, Pseudomonas, Salmonella. To different degrees the aminoglycosides are also active against Staphilococcus aureus, Staphilococcus epidermidis, Listeria and bacteria from the genera Mycobacteria.
Because aminoglycosides are highly polar cationic compounds, diffusion across the bacterial cell membrane is very limited and intracellular accumulation of the antibacterial agents is brought about by active transport. Many organisms display resistance to the older aminoglycosides. In addition, an increase in the resistance of microorganisms to the more recently introduced aminoglycosides is steadily rising. Increasing evidence suggests that acquired antibiotic resistance is often due to a balance between outer membrane penetration rate and the subsequent enzyme inactivation rate. Thus, the outer membrane barrier and the antibiotic-degrading enzymes are strongly synergistic. Moreover, while a newer aminoglycoside, by virtue of its insusceptibility to bacterial degrading enzymes, is active against strains resistant to older members of the group, can not be used to predict its activity in general, in view of the relative impermeability of a significant number of strains.
Although the aminoglycosides are useful for treating infections, their use can be accompanied by toxicity and side effects. The most important toxic effects are ototoxicity and nephrotoxicity. Because aminoglycosides can produce concentration-related oto-and nephrotoxicity, it is important to ensure that their plasma concentrations do not exceed toxic levels. It is equally important to ensure that fear of toxicity does not result in therapeutically inadequate dosage.
The encapsulation of aminoglycosides and .beta.-lactam antibiotics into liposomal formulations by the dehydration-rehydration vesicle (DRV) method has been described (Lagace et al., 1991, J. Microencapsulation 8:53-61). Disteroyl phosphatidyl-choline (DSPC) and dimyristoyl phosphatidyl-glycerol (DMPG), two synthetic phospholipids were used at a molar ratio 10:1 and at a lipid concentration of 16.5 umol/ml. The same liposomal formulation was tested "in situ" in an animal model of chronic pulmonary infection with Pseudomonas aeruginosa and permitted a marked increase of the residence time of antibiotic in lungs and a reduced systemic antibacterial agent absorption. Nevertheless, this liposomal aminoglycoside formulation did not show an improvement in the bactericidal activity as compared to free antibiotics and other controls (Omri et al., 1994, Antimicrob. Agents Chemother. 38:1090-1095). Other groups have disclosed aminoglycoside liposomal formulations (Da Cruz et al., 1993, WO 93/23015 and Proffitt et al., 1994, WO 94/12155). Nevertheless, the disclosed formulations fail to display a very drastic enhancement of the therapeutic activity of the antibiotic as compared to its activity in the free form. Indeed, the preferred aminoglycoside (netilmicin) liposomal formulation of Da Cruz et al., which comprises phosphatidylcholine (PC), cholesterol and phosphatidyl-inositol (PI), only shows a modest increase activity in vivo with the aminoglycoside as part of the liposomal formulation as compared to free aminoglycoside (at best by a factor of three). Proffitt et al., disclose a different aminoglycoside (amikacin) liposomal formulation comprising PC, cholesterol and distearoyl phosphatidylglycerol (DSPG). Although the Proffitt et al., formulation appears to be superior at enhancing the in vivo therapeutic activity of the aminoglycoside as compared to that of Da Cruz, this increase is still relatively low and dependent on the tissue (10-fold increase in spleen, 5-fold in liver and only 2-fold in lung). Importantly, the available liposomal formulations for use in treating bacterial infections do not appear to increase significantly the passage of the therapeutic agent through the bacterial membrane.
Cystic fibrosis (CF) is one of the most common lethal genetic diseases in humans. While the course of CF, varies greatly from patient to patient, it is largely determined by the degree of pulmonary involvement. In CF, deterioration appears unavoidable, and eventually leads to death. Although a CF patient prognosis has drastically improved in the second half of the century, the average survival is only 30 years of age. Of importance, a correlation between early colonization of Pseudomonas and a worse prognosis for CF patients has been observed. In addition, chronic lung infection due to Pseudomonas aeruginosa is the major cause of morbidity and mortality in patients wits cystic fibrosis (Omri et al., 1994, Antimicrob. Agents Chemother. 38:1090-1095; and Merck manual, 1992, 16th Edition, Merck Res. Lab.). In CF patients, Staphylococcus aureus, and Haemophilus influenza other Gram negative strains, are generally the early isolated pathogens. Such bacterial infections in CF patients are, in most cases, efficiently treated with antibiotics. A number of antibiotics are used for the antibacterial therapy, either alone or in combination. The choice of a particular antibiotic regimen depends on a number of factors which include the site and severity of the infection as well as the resistance/sensitivity profile of the microorganism. Of importance is the fact that high doses of antibiotics, especially aminoglycosides, as well as long-term antibiotic treatment are often indicated in CF patients.
Pseudomonas aeruginosa colonize more than 90% of CF adolescents. Efficient therapy targeted against Pseudomonas aeruginosa remains difficult and controversial (Omri et al., 1994, Antimicrob. Agents Chemother. 38:1090-1095). The usual standard therapy for CF patients colonized with this microorganism involves the use of an aminoglycoside or .beta.-lactam alone or in combination. These antibacterial agents require frequent high-dose parenteral administration in order to achieve therapeutically effective concentrations in serum, particularly against biofilm cells formed by the mucoid phenotype of P. aeruginosa. It should be noted that the outer-membrane (OM) permeability of P. aeruginosa is only about 1-8% that of E. coli, as assessed by antibiotic penetration rates (Yoshimura et al., 1982, J. Bacteriol. 152:636-642; Nicas et al., 1983, J. Bacteriol. 153:281-285; and Angus et al., 1982, Antimicrob. Agents Chemother. 21:299-309). It has also been reported that prolonged or repeated treatment with antibiotics has been associated with gradually decreasing susceptibility of this organism and with accelerated clearance of antibiotics in these patients (Omri et al., 1994, Antimicrob. Agents Chemother. 38:1090-1095; and references therein). Thus, although the use of liposomes as a vehicle for antibiotics, has been shown in "in vitro" experiments to be a promising avenue for the treatment of P. aeruginosa (Lagace et al., 1991, J. Microencapsulation 8:53-61; and Nacucchio et al., 1988, J. Microencapsulation 5:303-309), the design of a liposomal formulation permitting a significant improvement in the activity of the antibiotic as well as a significantly improved penetration inside the bacterial cell is yet to emerge. The design of such a liposomal formulation would be of tremendous importance in the treatment, and/or prophylaxis of bacterial infections in CF patients, and perhaps on the prognosis of these patients.
Although microorganism resistance to antibiotics has long been recognized, it continues to be an important health problem world-wide. Furthermore, based on the relative impermeability of numerous strains to antibiotics, the design of newer more efficient versions thereof, which can overcome the strain-based enzymatic degradation, still does not solve the significant hurdle of getting the antibiotic through the impermeable membrane or through an exopolysaccharide layer of the bacteria and to its site of action. Furthermore, the problem of increased resistance to antibiotics is compounded by the misuse of these agents (Merck manual, 1992, 16th Edition, Merck Res. Lab.). For example, because of the antibiotic resistance of microorganisms, which is more acute with older types of antibiotics, practitioners are often prompted to use a newer generation antibiotic, thereby contributing to the increased resistance of microorganisms to newer generation antibiotics. The large scale use of antibiotics in animals, including but not limited to dairy cows, and the presence of these antibiotics in milk or in the environment, is yet another contributor to the increase in microorganism resistance to antibiotics.
It would be of tremendous importance for the clinician to be able to increase the activity of antibiotics thereby potentially permitting a lowering of the doses required to attain the aimed anti-bacterial action. Furthermore, such increase in antibiotic activity would permit a more efficient use of older generation antibiotics, thereby moderating the increase in microorganism resistance to new generation antibiotics.
It would be a very significant advantage for the clinician, veterinarian or the like, to be able to use a liposome formulation containing an anti-bacterial agent, such as an antibiotic, wherein the liposomal formulation significantly improves the anti-bacterial activity of the agent, not only because of increased circulation time, and lower toxicity, but also because this formulation comprises phospholipids that markedly improve the penetration of the agent in a bacterial cell. It would further be of great advantage if the formulation also permitted a marked increase in the penetration of the anti-bacterial agent through the outer membrane (OM) and mucoid exopolysaccharides such as those secreted by mucoid variants of bacteria such as that of Pseudomonas aeruginosa.
In addition, it would be a tremendous advantage to have access to a therapeutic liposomal formulation, wherein the composition of the formulation permits modulated release of the therapeutic agent, over time thereby reducing side-effects and prolonging the action of the agent.